Oviductal fluid proteins have been shown to associate with gametes in several species. The objective of the present study was to identify bovine oviductal fluid proteins that associate with the bovine zona pellucida. Oviductal fluid was obtained daily from two dairy cows with normal oestrous cycles via indwelling oviductal cannulae. Fluid was collected from the ampullary and isthmic regions of the same oviduct. Oviductal fluid samples were pooled by oviduct region and according to stage of the oestrous cycle as determined by the concentration of serum progesterone. Samples collected when serum progesterone concentrations were > 1.5 ng ml−1 were combined into luteal pools. Non-luteal pools consisted of oviductal fluid samples collected on days when serum progesterone concentrations were ≤1.5 ng ml−1. Each oviductal fluid sample was assayed for protein concentration, and an aliquot equivalent to 10 mg ml−1 was biotinylated using biotinamidocaproate N-hydroxysuccinimide ester at a concentration of 1 mg ml−1. Cumulus-free, non-viable bovine oocytes were incubated in the biotinylated oviductal fluid samples for 3.5 h. Oocytes were washed, the zonae mechanically ruptured, solubilized, and subjected to one-dimensional SDS-PAGE. Separated proteins were transferred to nitrocellulose and probed with avidin–horseradish peroxidase. Five biotinylated oviductal fluid proteins were found to associate with the zona pellucida in all treatments. These proteins had apparent molecular masses of 80, 74, 60, 45, and 30 kDa. An additional protein, of molecular mass 95 kDa, was found associated with zonae from oocytes incubated in non-luteal fluid of both regions, but not from oocytes incubated in luteal fluid. This protein was shown to be bovine oestrus-associated protein by using a monospecific polyclonal antibody.
A. L. Staros and G. J. Killian
C. C. Wegner and G. J. Killian
Summary. The aim of the present study was to determine whether the synthesis of an oestrus-associated protein found in bovine oviductal fluid varies with oviductal region, stage of cycle or day of pregnancy. Explant culture was performed using oviducts recovered from naturally cycling animals either at oestrus or 12–14 days after oestrus. Three oviductal regions, the preampulla, ampulla and isthmus, were cultured individually in the presence of 20 μCi [35S]methionine in serum-free medium for 6 h at 37°C. Synthesis of oestrus-associated protein was assessed by one-dimensional SDS-PAGE, fluorography and densitometry of radiolabelled bands. Significantly more oestrus-associated protein was synthesized by the ampullar region of the oviduct, although it was detected in explant culture media from both the isthmic and preampullar regions. A polyclonal antibody produced against oestrus-associated protein was used to localize the protein in paraffin-embedded sections of oviductal explant cultures and other bovine tissues. Localization of the protein in oviductal tissue sections varied with stage of cycle (oestrus > luteal > pregnant) and region of oviduct (ampulla > preampulla/isthmus). These findings indicate an effect of oviductal region and hormonal state (cycling versus pregnant) on the synthesis and secretion of the oestrus-associated protein. Lectin affinity studies indicated that galactosyl(β1, 3)Nacetylgalactosamine and N-acetylglucosamine residues are associated with the oestrusassociated protein.
Keywords: oviductal fluid; protein synthesis; oestrous cycle; glycoprotein; cow
G. J. KILLIAN and R. P. AMANN
Hydroxysteroid dehydrogenases (HSD) were evaluated histochemically in tissues representing seven regions of the epididymides from twenty-five neopubertal and three adult bulls. The epithelium of the epididymides was devoid of HSD activity against each of twenty steroid substrates tested; only 3β,16β-dihydroxy-5-androsten 3-methyl ester was dehydrogenated. In luminal spermatozoa, however, 3α-, 3β-, 16β- and 17β-HSD activities were detected in sections from most neopubertal and all adult bull epididymides incubated with 5β-androsterone, 5β-epiandrosterone, 3β,16β-dihydroxy-5-androsten 3-methyl ester and 5β-dihydrotestosterone. All other substrate incubations were negative. There was no age-associated difference in demonstrable HSD activity of epididymal spermatozoa. In smears of unwashed testicular spermatozoa from adult bulls, 3α-HSD activity was most conspicuous on the sperm head and cytoplasmic droplet, 3β-HSD activity was most prevalent in the mid-piece and the 17β-HSD activity predominated in the mid-piece and the cytoplasmic droplet.
M. A. Henault and G. J. Killian
The objectives of this study were to determine the types of lipid synthesized and secreted by the bovine oviduct, and to determine whether lipid synthesis and secretion varied with stage of the ovarian cycle and oviductal region. Oviduct explant cultures were prepared from cows killed during either the follicular or luteal stage of the oestrous cycle. Both stage of ovarian cycle and oviductal region affected lipid synthesis by oviductal explants in vitro. More lipid was synthesized by explants from follicular than from luteal-stage cows. Ampullar explants synthesized the greatest quantity of total lipid, followed by the preampulla and isthmus. Separation of extracted lipids from cultured tissue by high performance thin-layer chromatography (HPTLC) resolved phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin, free cholesterol, free fatty acid, triglyceride and esterified cholesterol, all of which were synthesized during culture. The ampulla synthesized significantly more phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol than did the other regions. Culture supernatants from ampullary explants contained the most newly synthesized cholesterol when compared with other regions. The histochemical location of neutral lipid droplets in the epithelium of cultured explants paralleled the localization of radioactivity in autoradiographs of explant extracts. The results suggest that the oviduct synthesizes a variety of lipids, and that some of these are released into culture supernatants.
M. A. Henault and G. J. Killian
The ability of seminal plasma to influence the fertility of ejaculated bull spermatozoa was examined using a sperm penetration assay for zona-free bovine oocytes. Washed, ejaculated spermatozoa from bulls of below (low) or above average (high) fertility were mixed with seminal plasma from the same bull, or with seminal plasma from a bull of contrasting fertility. Treated spermatozoa were stained with different fluorochromes and competed to penetrate oocytes after heterospermic insemination in vitro. Washed spermatozoa exposed to seminal plasma from bulls of high fertility penetrated more oocytes than those spermatozoa mixed with seminal plasma from bulls of low fertility (P < 0.01). Mixing low fertility spermatozoa with high fertility seminal plasma generally improved penetrating ability compared with low fertility spermatozoa mixed with low fertility seminal plasma (P = 0.05). Washed spermatozoa from a bull of low fertility mixed with his own seminal plasma had greater ability to penetrate oocytes than did washed spermatozoa from a bull of high fertility mixed with seminal plasma from a bull of low fertility (P < 0.01). The bias associated with using low fertility seminal plasma from the bull providing the spermatozoa was removed by repeating this experiment using pooled seminal plasma from different subfertile bulls. After combination with pooled seminal plasma from bulls of low fertility spermatozoa from bulls of high or low fertility penetrated oocytes in a similar way, but high fertility spermatozoa had a slightly higher penetration rate (P = 0.12). In conclusion, the penetration of zona-free oocytes by ejaculated spermatozoa from bulls of low fertility was marginally improved by seminal plasma from bulls of high fertility, but penetration by high fertility spermaotoza was decreased by exposure to low fertility seminal plasma. Seminal plasma from bulls of low fertility similarly affected the penetrating ability of high and low fertility spermatozoa if the seminal plasma used was foreign to the spermatozoa being tested.
G. W. Robb, R. P. Amann and G. J. Killian
Summary. Rats were considered to be pubertal at 50 days of age when spermatozoa were first found in the tail of the epididymis. Sperm production/g testis increased up to 75 days of age and testicular weight increased until 100 days of age. Sperm reserves in the tail of the epididymis were not maximal until 100 days of age. Therefore, Wistar rats are not sexually mature until 100 days. Sexually mature rats had testes weighing 3·7 g, produced 86 × 106 spermatozoa or 24 × 106 spermatozoa/g testicular parenchyma daily, and their paired epididymides contained 295 × 106 spermatozoa in the head + body and 440 × 106 spermatozoa in the tail.
A. A. Grippo, A. L. Way and G. J. Killian
Motility, acrosome reaction and oocyte fertilizing ability were assessed for bull spermatozoa after incubation in regional (isthmic or ampullary), bovine oviductal fluid, pooled by stage of the oestrous cycle. Oviductal fluids collected daily from isthmic and ampullary cannulae implanted in the same oviduct were divided into pools, representing two oestrous cycle stages, based on daily serum progesterone concentrations. Ejaculated bull spermatozoa were incubated for 0–6 h in each type of oviductal fluid. Incubation in isthmic oviductal fluid collected during the nonluteal stage, including oestrus and ovulation, decreased overall sperm motility (from 71.7% motile spermatozoa to 34.0% and both path (78 μm s−1 versus 86–89 μm s−1) and progressive (74 μm s−1 versus 83–85 μm s−1) velocities of spermatozoa motion. Spermatozoa incubated in isthmic, non-luteal oviductal fluid had a higher rate and extent of sperm acrosome reaction (213% of control versus 136–161% of control by 2 h incubation) compared with spermatozoa incubated in other oviductal fluid types. However, incubation in nonluteal ampullary fluid increased the number of spermatozoa, which were both acrosome reacted and live, and able to fertilize bovine ova (88.7% fertilized versus 75–81%). Glycosaminoglycan concentrations were similar among types of oviductal fluid (0.77–0.88 mg ml−1). These findings indicate that oviductal fluid differentially affects sperm function, depending on the oviduct region and the stage of the oestrous cycle at which the fluid was obtained.
R. P. AMANN, G. J. KILLIAN and A. W. BENTON
Polyacrylamide gel electrophoresis was used to evaluate rete testis fluid (RTF) and plasma from the cauda epididymidis (CEP) obtained from conscious Holstein bulls. The major protein in RTF had an electrophoretic mobility similar to that of blood serum albumin, but the seemingly analogous protein in CEP had a slower relative mobility. Bovine CEP contains at least three proteins, one glycoprotein and possibly three other PAS-positive components not detected in RTF or blood serum. In addition, esterases, acid phosphatases and several β-glucuronidases have been detected in CEP which were absent in RTF. Agarose immunoelectrophoretic analyses confirmed the basic differences among the three fluids. Although RTF and CEP share certain proteins with blood serum, each fluid is biologically unique. Certain proteins detected in CEP but not in RTF or blood serum probably originated from the testicular spermatozoa while others may represent epididymal secretion.
A. L. Way, A. M. Schuler and G. J. Killian
The objective of this study was to determine the effects of oviductal fluid on sperm binding and fertilization in vitro when either bovine oocytes or both spermatozoa and oocytes are incubated in fluid from different regions of the oviduct and at different stages of the oestrous cycle. Oocytes, or oocytes and spermatozoa, were incubated in nonluteal and luteal oviductal fluid obtained from the whole, isthmic or ampullary oviduct of dairy cows. Gametes were washed and coincubated in fertilization medium for 18 h, and then observed for sperm binding and penetration. More spermatozoa bound to oocytes that were preincubated in nonluteal oviductal fluid than to those that were incubated in luteal oviductal fluid; however, there were no differences in fertilization rates between the two treatments. More spermatozoa bound to oocytes incubated in isthmic than in ampullary oviductal fluid. However, if both gametes were incubated in oviductal fluid before their coincubation, binding was higher when both gametes were incubated in ampullary oviductal fluid. By reducing the time of coincubation of spermatozoa with the oocytes, differences in fertilization rates were seen among the various oviductal fluid treatments. At 14 and 16 h of coincubation, more oocytes were fertilized if spermatozoa were preincubated in isthmic oviductal fluid and oocytes were preincubated in ampullary oviductal fluid than if both gametes were incubated in whole or ampullary oviductal fluid. Because both sperm binding to the zona pellucida and fertilization rates were greater in treatments in which gametes were exposed to regional oviductal fluid than when they were incubated in whole oviductal fluid, it is concluded that oviductal fluid from different regions of the bovine oviduct may play different roles in facilitating fertilization.
G. J. Killian, D. A. Chapman, J. F. Kavanaugh, D. R. Deaver and H. B. Wiggin
Summary. The oviducts of 4 cows were cannulated and oviduct fluid was collected daily from the exteriorized cannulas for a total of 5 oestrous cycles. Daily serum samples were assayed for oestradiol-17β and progesterone to monitor the oestrous cycle. Data for each cycle were compared for oviduct fluid collected during the non-luteal phase (serum progesterone ⩽ 1·5 ng/ml) and the luteal phase (serum progesterone > 1·5 ng/ml). During the non-luteal phase oviduct fluid volume was higher and the osmolality was lower than during the luteal phase. Total protein, cholesterol and phospholipid secreted daily was greater during the non-luteal phase. Cholesterol and protein concentrations were generally lower during the non-luteal phase, but phospholipid concentrations were generally higher. About 40% of the phospholipid in oviduct fluid was phosphatidylcholine and lysophosphatidylcholine, while phosphatidylinositol and lysophosphatidylinositol accounted for 20%. The ratio of 1-acyl-phospholipid to diacylphospholipid increased during the non-luteal phase. An increased cholesterol to phospholipid ratio, and a decreased cholesterol to protein ratio in oviduct fluid also were associated with the non-luteal phase. Changes in the lipid composition of oviduct fluid during the oestrous cycle may play a role in the preparation of gametes for fertilization.
Keywords: oviduct fluid; cow; phospholipid; cholesterol; protein